*Target (protein/gene name): tyrosine-phosphatase *NCBI Gene # or RefSeq#:FR687359.1 *Protein ID (NP or XP #) or Wolbachia#:
YP_004029639.1
1U2P *Organism (including strain): Mycobacterium Tuberculosis Etiologic Risk Group (see link below): *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium Tuberculosis is the causative agent of most cases of tuberculosis. Tuberculosis is a common, infectious disease that is oftentimes fatal. It normally attacks the lungs, but can attack other parts of the body too. It's can be spread by an infectious person coughing or sneezing and another person being within the vicinity of the virus in the air. People who have HIV and live in poverty stricken areas are more susceptible to contracting the disease. Most infections don't show sypmtoms, but when they do, they may include a chronic cough, fever, night sweats, and weight loss. Its current main method of prevention is through vaccination and informing people about the risks of the disease. Essentiality of this protein: essential Complex of proteins?: no Druggable Target: yes
3. 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP)
$208 per 20UG
Protein tyrosine phosphatase. (PTP-IA2, ICA-512), Catalytic domain, (amino acids 693- 979), (Gene bank accession no. NM_002846), with N-terminal GST tag, MW = 58.8 kDa, expressed in an E. coli expression system. http://www.sigmaaldrich.com/catalog/product/sigma/srp0221?lang=en®ion=US
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1U2P
Current Inhibitors: ALN, orthovanadate, and PAO Expression Information (has it been expressed in bacterial cells):
It has a high yield for bacterial expression (~1000 mg/L of TB) but the expressed protein was only found in insoluble precipitates. The most often used method for expression is by insect cell culture. Purification Method:
The protein is purified under denaturing conditions and yields active kinase.
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTD HRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFA VIESALPGLHDWVDERLARNGPS
*length of your protein in Amino Acids: 163 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17.892 Molar Extinction coefficient of your protein at 280 nm wavelength: 15595 M-1 cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only): GTGTCTGATCCGCTGCACGTCACATTCGTTTGTACGGGCAACATCTGCCGGTCGCCAATGGCCGAGAAGA TGTTCGCCCAACAGCTTCGCCACCGTGGCCTGGGTGACGCGGTGCGAGTGACCAGTGCGGGCACCGGGAA CTGGCATGTAGGCAGTTGCGCCGACGAGCGGGCGGCCGGGGTGTTGCGAGCCCACGGCTACCCTACCGAC CACCGGGCCGCACAAGTCGGCACCGAACACCTGGCGGCAGACCTGTTGGTGGCCTTGGACCGCAACCACG CTCGGCTGTTGCGGCAGCTCGGCGTCGAAGCCGCCCGGGTACGGATGCTGCGGTCATTCGACCCACGCTC GGGAACCCATGCGCTCGATGTCGAGGATCCCTACTATGGCGATCACTCCGACTTCGAGGAGGTCTTCGCC GTCATCGAATCCGCCCTGCCCGGCCTGCACGACTGGGTCGACGAACGTCTCGCGCGGAACGGACCGAGTT GA
*GC% Content for gene:
66% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: FR687359.1
*Protein ID (NP or XP #) or Wolbachia#:
YP_004029639.1
1U2P
*Organism (including strain): Mycobacterium Tuberculosis
Etiologic Risk Group (see link below):
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium Tuberculosis is the causative agent of most cases of tuberculosis. Tuberculosis is a common, infectious disease that is oftentimes fatal. It normally attacks the lungs, but can attack other parts of the body too. It's can be spread by an infectious person coughing or sneezing and another person being within the vicinity of the virus in the air. People who have HIV and live in poverty stricken areas are more susceptible to contracting the disease. Most infections don't show sypmtoms, but when they do, they may include a chronic cough, fever, night sweats, and weight loss. Its current main method of prevention is through vaccination and informing people about the risks of the disease.
Essentiality of this protein: essential
Complex of proteins?: no
Druggable Target: yes
*EC#: 3.1.3.48
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48&Suchword=&organism[]=Mycobacterium+tuberculosis&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates):
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/5/t1196dat.Par.0001.File.tmp/t1196dat.pdf
-- link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pubmed/15588980
-- List cost and quantity of substrate reagents and supplier:
1. p-nitrophenyl phosphate (pNPP) - Phosphatase Inhibitor Cocktail 3DMS
$61.40 per 1ML
Recommended dilution is 1 mL of the cocktail per 500 mg of protein extracted from the tissue or cells.
http://www.sigmaaldrich.com/catalog/product/sigma/p0044?lang=en®ion=US
2. fluorescein diphosphate (FDP)
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/7/f2678dat.Par.0001.File.tmp/f2678dat.pdf
3. 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP)
$208 per 20UG
Protein tyrosine phosphatase. (PTP-IA2, ICA-512), Catalytic domain, (amino acids 693- 979), (Gene bank accession no. NM_002846), with N-terminal GST tag, MW = 58.8 kDa, expressed in an E. coli expression system.
http://www.sigmaaldrich.com/catalog/product/sigma/srp0221?lang=en®ion=US
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1U2P
Current Inhibitors: ALN, orthovanadate, and PAO
Expression Information (has it been expressed in bacterial cells):
It has a high yield for bacterial expression (~1000 mg/L of TB) but the expressed protein was only found in insoluble precipitates. The most often used method for expression is by insect cell culture.
Purification Method:
The protein is purified under denaturing conditions and yields active kinase.
Reference for protein purification and expression:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253236/
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAAGVLRAHGYPTD HRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSGTHALDVEDPYYGDHSDFEEVFA VIESALPGLHDWVDERLARNGPS
*length of your protein in Amino Acids: 163
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 17.892
Molar Extinction coefficient of your protein at 280 nm wavelength: 15595 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GTGTCTGATCCGCTGCACGTCACATTCGTTTGTACGGGCAACATCTGCCGGTCGCCAATGGCCGAGAAGA TGTTCGCCCAACAGCTTCGCCACCGTGGCCTGGGTGACGCGGTGCGAGTGACCAGTGCGGGCACCGGGAA CTGGCATGTAGGCAGTTGCGCCGACGAGCGGGCGGCCGGGGTGTTGCGAGCCCACGGCTACCCTACCGAC CACCGGGCCGCACAAGTCGGCACCGAACACCTGGCGGCAGACCTGTTGGTGGCCTTGGACCGCAACCACG CTCGGCTGTTGCGGCAGCTCGGCGTCGAAGCCGCCCGGGTACGGATGCTGCGGTCATTCGACCCACGCTC GGGAACCCATGCGCTCGATGTCGAGGATCCCTACTATGGCGATCACTCCGACTTCGAGGAGGTCTTCGCC GTCATCGAATCCGCCCTGCCCGGCCTGCACGACTGGGTCGACGAACGTCTCGCGCGGAACGGACCGAGTT GA
*GC% Content for gene:
66%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48&Suchword=&organism[]=Mycobacterium+tuberculosis&show_tm=0
http://www.rcsb.org/pdb/explore/explore.do?structureId=1U2P
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/5/t1196dat.Par.0001.File.tmp/t1196dat.pdf